Diagnostic markers for ischemia

ABSTRACT

This invention relates to the diagnosis and monitoring of ischemia, including but not limited to myocardial and cerebral ischemia, by measuring the concentration of molecules that do not originate from the ischemic tissue but whose concentration in the blood and other fluids changes as a consequence of the ischemic state.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/322,523, filed Sep. 14, 2001, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the diagnosis and monitoring of ischemia,including but not limited to myocardial and cerebral ischemia, bymeasuring the concentration of molecules that do not originate from theischemic tissue but whose concentration in the blood and other fluidschanges as a consequence of the ischemic state.

2. Description of the Related Art

Ischemia is a reduction in blood flow. This reduction may occur for avariety of reasons, including but not limited to thrombosis, embolism,aneurysm, spasm, or collapse of a blood vessel due to deterioration.Because of the reduction in blood flow the tissue that would otherwisebe nourished may no longer receive sufficient nutrition to maintaincellular integrity, it also may not be able to remove sufficient amountsof cellular waste products and it may also result in inadequate exchangeof blood gases such as oxygen and carbon dioxide. The inability totransfer sufficient oxygen to the cells (hypoxia) may have many of thesame consequences of ischemia and can also be detected by the samemethods claimed in this invention.

If ischemia persists for a sufficient time, that is, if oxygenated bloodflow is not restored, then the cells of the tissue normally perfused bythe blood flow, will begin to die. This may occur gradually over timeand may be unnoticed until sufficient cell destruction has occurred sothat the function of the organism is significantly impaired. An examplemay be the gradual deterioration of circulation to the extremities orother body parts that occurs in diabetes. The disruption of blood flowmay also occur more acutely. This includes but is not limited tothrombus formation that results in reduction of blood flow through thecoronary arteries, lodging of an embolism in a cerebral ischemia andsimilar events in the kidney and limbs.

For either a gradual or an acute time course, the earlier theischemic/hypoxic condition can be detected and the sooner palliativetherapy can be applied, the better will be the outcome for tissue andorganism. For example, early detection of the diabetic-mediated ischemiain extremities might avoid amputation and early detection and relief ofacute blockages to the heart or brain significantly reduces mortalityand morbidity.

Unfortunately, early detection of ischemia/hypoxia is often notpossible. For example, the ECG which is the primary early diagnostictool for acute coronary syndromes is less than 40% sensitive. For strokepatients the only tools available are either a CT scan or MRI, both ofwhich can only determine if the patient has a hemorrhage in the firstseveral hours after symptoms began. Only much later, when it is too lateto administer the only treatment of ischemic stroke, thrombolytictherapy, are these imaging methods able to determine if an ischemicstroke has occurred. Thrombolytic therapy must be administered within 3hours of symptoms. For other organs there are no well establishedmethods for early detection of ischemia. Thus a sensitive, accurate andrapid test for ischemia is needed for the diagnosis and treatment ofpatients.

In the final stages of ischemia, when cells begin to die (necrosis),they may release some of their contents into the blood. These areprimarily intracellular proteins that are released because the normalbarrier to containment, the cell's membrane, is compromised bybiochemical changes associated with death. Often these molecules aretissue specific, for example, cardiac troponin in the case of the heart.Although these molecules accurately reflect the presence of disease,they generally require several hours after symptoms occur to reachlevels of significance in blood and they are only released from dead ordying cells. Thus in addition to sensitivity and accuracy an importantfeature of a test for ischemia would be its ability to detect theischemic state well before necrosis.

In addition to the release of molecules from the ischemic tissue thatare markers of necrosis, the ischemic event may generate a series ofbiochemical changes that can result in the change in concentration ofmolecules within the blood or other fluids that do not originate fromthe ischemic tissue. These are referred to in this invention as ischemicmarkers. The generation of ischemic markers can occur, for example, whenthe ischemic tissue generates molecules that are then converted todifferent molecules by a non-ischemic tissue or the ischemic tissuegenerates molecules that activate, from non-ischemic tissue, the releaseof different molecules into body fluids. Examples of this are thegeneration of norepinephrine, TNF_(α) and natriuretic peptides byischemic cardiac or cerebral tissue. The norepinephrine, TNF_(α) andnatriuretic peptides so generated activate lipolysis in adipose tissueresulting in the elevation of free fatty acids in blood. In anotherexample, sphingosine released from the ischemic myocardium is convertedto sphingosine-1-phosphate in platelets and may then be detected inblood (Yatomi, et al. (1997) Journ. Biol. Chem. vol. 272: pages5291-5297; U.S. Pat. No. 6,210,976).

In addition to diagnosing the presence of disease, levels of ischemicmarkers may predict risk of future deleterious events. Moreover, thesemolecules, at sufficient levels, may themselves mediate cellular effectsthat result in deleterious outcomes. For example, a large long termstudy of apparently healthy men revealed that increasing levels of totalserum free fatty acids (FFA), although within the normal range, wereassociated with an increased risk of sudden death 22 years later. It wasspeculated that this increased rate of death was a consequence of FFAinduced cardiac arrhythmias. In another example, use of the combinationof glucose-insulin-potassium (GIK) in patients suffering from acutemyocardial infarcts, produced a significant reduction in mortalityrelative to patients who did not receive GIK. One theory for thisbeneficial effect is the reduction in serum total FFA produced by GIK.Thus there is a need to be able to monitor ischemic markers to evaluatelonger term risk of disease and to help to decide on the type oftherapeutic intervention to reduce the increased risk associated withwhat may be a chronic ischemic state.

SUMMARY OF THE INVENTION

The present invention describes the use in the diagnosis and monitoringof ischemia, of measuring the concentration in blood and/or other fluidsof molecules that do not originate from the ischemic tissue. Thesemolecules are called ischemic markers. The change in the concentrationof these ischemic markers, relative to the non-ischemic condition, isindicative of some type of ischemia.

In one embodiment, the present invention is drawn to a method ofdetecting a condition which is indicative of ischemia in a mammal whichincludes the steps of:

-   (a) measuring a level of an ischemic marker in a test sample from a    fluid of the mammal; and-   (b) determining if the level of the ischemic marker measured in the    test sample correlates with ischemia in the mammal. In a preferred    embodiment, the mammal is a human. The fluid used for the test    sample may be selected from the group including but not limited to    blood, serum, plasma, saliva, bile, gastric juices, cerebral spinal    fluid, lymph, interstitial fluid or urine. In a preferred    embodiment, the fluid used for the test sample is blood.

In a one embodiment, the ischemic marker is a lipid. The lipid may beselected from the group including, but not limited to, a sphingolipid, alysolipid, a glycolipid, a steroid, and an eicosanoid, includingleukotrienes, prostacyclins, prostaglandins and thromboxanes. In oneembodiment, the sphingolipid used as the ischemic marker may besphingosine, or a metabolite thereof including ceramide (Cer,N-acylsphingosine), sphingosine-1-phosphate,sphingosylphosphorylcholine, or dihydrosphingosine, for example. In amore preferred embodiment, the lipid is a fatty acid.

The disclosed method may be used for detection of ischemia in any organor tissue including but not limited to the heart, brain, kidney or alimb.

In one embodiment of the invention, a component of the ischemic markerwhich is soluble in an aqueous buffer is detected. In an alternateembodiment, the component of the ischemic marker which is not soluble inan aqueous buffer is detected.

The ischemic marker may be detected by spectroscopic means including butnot limited to UV/VIS, infrared, microwave, radio, absorption oremission spectroscopes. Chromatographic procedures are also encompassedby the present invention, including but not limited to HPLC, lowpressure chromatography, medium pressure chromatography, and gaschromatography. Detection of the ischemic marker by electron spinresonance using a spin label is also encompassed by the invention. In analternate embodiment, the ischemic marker may be detected by an antibodyor receptor molecule, immunoassay or enzymatic assay.

In a preferred embodiment, the method may include an initial step ofselecting a mammal presenting symptoms of ischemia. In some embodiments,the method may also include the step of administering anti-ischemictherapy if the level of ischemic marker correlates with ischemia in saidmammal. The anti-ischemic therapy may include a means to lower levels ofserum fatty acids. In some embodiments, the anti-ischemic therapy isreperfusion therapy, antithrombolytic therapy, angiogenic therapy orsurgery.

In some embodiments, the determination step is a comparison between saidmeasured level of said ischemic marker and a predetermined value for thelevel of said marker. In some embodiments, the predetermined value forthe level of the marker is indicative of the non-ischemic condition.

In one embodiment, the ischemic marker is an unbound or water-solublefree fatty acid. In a preferred embodiment, the unbound or water-solublefree fatty acid is detected by a protein that binds fatty acid. In apreferred embodiment, the protein that binds an unbound or water-solublefree fatty acid is fluorescent and exhibits a fluorescence that isdifferent when the fatty acid is bound than when it is not bound. Insome embodiments, the protein is one of the family of intracellularFatty Acid Binding Proteins (FABPs) that have molecular weights betweenabout 13,000 and 16,000 Dalton. In a preferred embodiment, the FABP is arat intestinal FABP. In a preferred embodiment, the FABP is covalentlylabeled with acrylodan at position 27. In a more preferred embodiment,the acrylodan-labeled FABP is the leucine 72 to alanine mutant.

In some embodiments, a level of the unbound or water-soluble free fattyacid greater than 2 standard deviation units above an average value of alevel of the unbound or water-soluble free fatty acid determined from anon-ischemic population is indicative of the ischemic condition. In someembodiments, a level of the unbound or water-soluble free fatty acidgreater than about twice an average value of a level of the unbound orwater-soluble free fatty acid determined from a non-ischemic populationis indicative of the ischemic condition. In some embodiments, a level ofthe unbound or water-soluble free fatty acid greater than about 5 nM isindicative of the ischemic condition.

In an alternate embodiment, the protein that binds an unbound orwater-soluble free fatty acid is albumin. In a preferred embodiment, thealbumin is covalently labeled with 7-hydroxycoumarin or anthraniloyl.

In an alternate embodiment, the ischemic marker is total free fattyacid. In an alternate preferred embodiment a level of the total freefatty acid greater than 2 standard deviation units above an averagevalue for the level of the total free fatty acid determined from anon-ischemic population is indicative of the ischemic condition. In analternate preferred embodiment, a level of the total free fatty acidgreater than about twice an average value of a level of the total freefatty acid determined from a non-ischemic population is indicative ofthe ischemic condition. In another embodiment, the ischemic marker is aratio of total free fatty acid to albumin.

In some embodiments, the ratio of total free fatty acid to albumingreater than about two standard deviation units above an average valueof the ratio of total free fatty acid to albumin determined from anon-ischemic population is indicative of the ischemic condition. In someembodiments, the ratio of total free fatty acid to albumin greater thanabout twice an average value of the ratio of total free fatty acid toalbumin determined from a non-ischemic population is indicative of theischemic condition.

In one embodiment, a method of determining patient response to atreatment for ischemia is described which includes the steps ofdetecting ischemia as described above and determining if the level ofthe ischemic marker is trending towards the level of the marker in thenon-ischemic condition. In one embodiment, the treatment is reperfusiontherapy which includes but is not limited to angioplasty oradministration of a thrombolytic agent.

In one embodiment, a method of identifying patients at high risk forhemorrhage after receiving reperfusion therapy is described whichincludes the steps of:

-   measuring the level of a lipid component in a body fluid sample from    the patient;-   comparing the measured level of the lipid component from the patient    to a threshold level of a lipid component, wherein the threshold    level is determined from measuring a lipid component in body fluid    of a normal population that does not have ischemia;-   determining a ratio of the measured level of the lipid component    from the patient to the threshold level; and-   correlating the ratio with the relative risk for hemorrhage after    reperfusion therapy such that a high ratio indicates a high risk. In    a preferred embodiment, the lipid component is selected from the    group including unbound free fatty acid, total free fatty acid and a    ratio of total free fatty acid to albumin.

In one embodiment, a method of identifying patients at high risk formortality within three years after an ischemic event is described whichincludes the steps of:

-   measuring the level of a lipid component in a body fluid sample from    the patient;-   comparing the measured level of the lipid component from the patient    to a threshold level of a lipid component, wherein the threshold    level is determined from measuring a lipid component in body fluid    of a normal population that does not have ischemia;-   determining a ratio of the measured level of the lipid component    from the patient to the threshold level; and-   correlating the ratio with the relative risk for mortality within    three years after an ischemic event such that a high ratio indicates    a high risk. In a preferred embodiment, the lipid component is    selected from the group including unbound free fatty acid, total    free fatty acid and a ratio of total free fatty acid to albumin.

In one embodiment, a method of identifying patients at high risk forhemorrhage is described which includes the steps of:

-   measuring a lipid component in a body fluid sample from the patient;-   comparing the measured level of the lipid component from the patient    to a threshold level of a lipid component, wherein the threshold    level is determined from measuring a lipid component in body fluid    of a normal population that does not have ischemia;-   determining a ratio of the measured level of the lipid component    from the patient to the threshold level; and-   correlating the ratio with the relative risk for hemorrhage such    that a high ratio indicates a high risk. In a preferred embodiment,    the lipid component is selected from the group including unbound    free fatty acid, total free fatty acid and a ratio of total free    fatty acid to albumin.

In another embodiment of the invention, a method is described fortreating patients at high risk for hemorrhage after receivingreperfusion therapy including the steps of:

-   identifying a patient at high risk for hemorrhage after reperfusion    therapy using the method described above; and-   treating said high-risk patient with an anti-ischemic therapy. In    one embodiment, the anti-ischemic therapy is selected from the group    including glucose-insulin-potassium compositions and albumin    compositions.

In another embodiment a method of treating patients at high risk formortality within three years after an ischemic event is described whichincludes the steps of:

-   identifying a patient at high risk for mortality within three years    after an ischemic event using the method described above; and-   treating the high-risk patient with an anti-ischemic therapy. In one    embodiment, the anti-ischemic therapy is selected from the group    including glucose-insulin-potassium compositions and albumin    compositions.

In another embodiment, a method of treating patients at high risk forhemorrhage is described which includes the steps of:

-   identifying a patient at high risk for hemorrhage using the method    described above; and-   treating the high-risk patient with an anti-ischemic therapy. In one    embodiment, the anti-ischemic therapy is selected from the group    including glucose-insulin-potassium compositions and albumin    compositions.

In one embodiment, a method of assessing long-term risk in non-acutepatients is described which includes the steps of:

-   measuring the level of a lipid component in a body fluid sample from    the patient;-   comparing the measured level of the lipid component from the patient    to a threshold level of a lipid component, wherein the threshold    level is determined from measuring a lipid component in body fluid    of a normal population that does not have ischemia;-   determining a ratio of the measured level of the lipid component    from the patient to the threshold level; and-   correlating the ratio with the relative risk for mortality such that    a high ratio indicates a high risk. In a preferred embodiment, the    lipid component is selected from the group including unbound free    fatty acid and a ratio of total free fatty acid to albumin.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 shows the level of unbound free fatty acid in blood samples takenfrom the Thrombolysis in Myocardial Infarction II (TIMI II) study ofpatients with AMI. Patients included in the study presented to theEmergency Department within 4 hours of onset of symptoms. Histogramrepresentation shows the results of [FFA_(μ)] measurements in blooddraws at times 0 (time of admission into Emergency Dept.), 50 min., 5hour and 8 hour for each of the first 250 patients, measured withADIFAB2.

FIG. 2 shows FFA_(u) results in the TIMI II study of patients with AMI.Histogram representation shows the results for the second set of 250patients measured with ADIFAB2. The results are virtually identical tothose in FIG. 1 for the first 250 patients.

FIG. 3 shows creatine kinase (CK) levels in TIMI II patients at time 0(admission time) (closed box, -▪-) and at 4 hours (closed triangle,-▴-). Values were determined by the TIMI investigators for patientscorresponding to shipments 1 and 2. The upper limit of normal wasdetermined for each day's measurements and for each study site (openbox, --□--).

FIG. 4 shows mortality increases with [FFA_(u)] values at admission inTIMI II patients. About 400 of the 500 patients in this set had blooddraws at time of admission. The levels of FFA_(u) were sorted and thenumber of deaths in each quartile were determined. The results yield apositive correlation that increases by more than 2 fold from lowest tohighest quartile (p<0.042). Median [FFA_(u)] for each quartile are shownin the bars.

FIG. 5 shows the correlation between [FFA_(u)] at admission and severehemorrhagic events in TIMI II patients. The number of severe hemorrhagicevents per quartile of FFA_(u) levels is shown. Numbers in the bars arethe median [FFA_(u)] in nM. The results indicate a more than 3 foldincrease in risk from lowest to highest quartile with a p<0.01.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the described embodiment represents the preferred embodiment ofthe present invention, it is to be understood that modifications willoccur to those skilled in the art without departing from the spirit ofthe invention. The scope of the invention is therefore to be determinedsolely by the appended claims.

The present invention describes the use, in the diagnosis and monitoringof ischemia, of measuring the concentration of molecules that do notoriginate from the ischemic tissue in blood and/or other fluids. Thesemolecules are called ischemic markers. The change in the concentrationof these ischemic markers, relative to the non-ischemic condition, isindicative of some type of ischemia. A marker is considered not tooriginate from the ischemic tissue even though some fraction may have sooriginated, so long as that fraction represents only a small portion ofthe marker present in the sample, e.g., 20%, 10%, 5%, 2%, 1% or less.

In one embodiment, the ischemic marker is a lipid component such as afatty acid. Total fatty acids may be determined by methods well known inthe art such as taught in U.S. Pat. Nos. 4,071,413, 5,512,429,5,449,607, and 4,369, 250 all of which are incorporated herein byreference. Levels of total fatty acid obtained by these methods may becompared with levels obtained from normal individuals in order to detectan ischemic condition in an individual. In a preferred embodiment, aratio of total fatty acid to albumin is determined and compared with anormal population.

In another embodiment, unbound free fatty acid levels are measured in abodily fluid as described below. Unbound free fatty acids (FFA_(u)) arethe portion of free fatty acids soluble in the aqueous phase. In mostbody fluids free fatty acid (FFA) is mostly bound to proteins, forexample, albumin, and membranes but a significant minority is unbound(FFA_(u)) and soluble in the aqueous phase. FFA_(u) are also referred toas water-soluble free fatty acids.

The bodily fluid may be selected from the group including cerebralspinal fluid, blood, serum, plasma, urine, saliva, lymph, gastricjuices, interstitial fluid or bile In preferred embodiments, the fluidused for the test sample is taken from blood.

In one embodiment, any assay that provides an indication of the level ofunbound free fatty acid (FFA_(u)) in body fluid relative to anasymptomatic population may be used in the diagnostic method to detectan ischemic condition. Preferably a threshold value is determined from anormal population that does not have an ischemic condition. In oneembodiment, the threshold value is a concentration of FFA_(u) in a bodyfluid that is significantly higher than the concentration of FFA_(u) inthe body fluid of a control population that does not have an ischemiccondition. In one embodiment, the threshold value is a concentration ofFFA_(u) in a body fluid that is at least about two standard deviationsgreater than an average concentration of FFA_(u) in a body fluid of acontrol population that does not have an ischemic condition. In anotherembodiment, the threshold concentration of FFA_(u) in a body fluid is atleast about 5 nM. In another embodiment, the threshold concentration ofFFA_(u) in a body fluid is at least about twice the averageconcentration of FFA_(u) in a body fluid of a control population thatdoes not have an ischemic condition.

The present methods are particularly valuable when applied to selectedpatient populations. Thus, in one embodiment, the method is applied to asample from an individual at risk for ischemia, such as diabeticpatients, or surgery patients, or patients with familial or lifestylerisk factors. In another embodiment, the method is applied to a patientpresenting symptomology consistent with ischemia or an ischemic event,or a patient suspected of having ischemia. Particular patientsubpopulations that can be selected include patients having symptomologyconsistent with cardiac ischemia, brain ischemia, kidney ischemia, limbischemia, or other clinically-significant ischemia. The method may beselectively applied to patients presenting symptomology consistent withor inconsistent with one particular type of ischemia. Non-limitingexamples would be selection of a patient having ischemic symptoms otherthan cardiac ischemia, or symptoms other than brain ischemia, orsymptoms other than peripheral or diabetic-related ischemia. The presentmethod may similarly be used to detect ischemia in cardiac patients inthe absence of myocardial infarction by selecting an appropriate patientpopulation. In one embodiment of any of the methods of the presentinvention, the patient is a mammal, and in a particular embodiment, thepatient is a human.

In still a further embodiment of the invention, the method of thepresent invention further includes a treatment step. Thus, a patient isfirst tested for an ischemic condition, represented by elevation of oneof the markers discussed herein. Patients having elevated levels of themarker are then treated with anti-ischemic therapy of any suitable type,such as reperfusion therapy, antithrombolytic therapy, angiogenictherapy, surgery, or the like. For example, patients may advantageouslybe tested within 10, 7, 5, or 3 hours of an ischemic event or symptom,and may then receive anti-ischemic therapy within 3, 2, or preferably 1hour of the test.

In one embodiment, the present invention uses a fluorescently-labeledfatty acid binding protein (FABP) to measure an increased amount ofunbound free fatty acid (FFA_(u)) in body fluid samples associated withan ischemic condition by quantitatively detecting a shift influorescence associated with binding of a FFA_(u) molecule to thefluorescently-labeled FABP. In serum, free fatty acid (FFA) is mostlybound to albumin but a significant minority is unbound (FFA_(u)) andsoluble in the aqueous phase. This invention utilizes the methodsubstantially as described in U.S. Pat. No. 5,470,714, which isincorporated herein by reference. A variety of FABP and fluorescentlabels can be used in detecting levels of FFA_(u) in body fluids such asblood. These include, but are not limited to, rat intestinal FABP(I-FABP), human adipocyte FABP (A-FABP) and rat heart FABP (H-FABP).Site-specific mutant forms of these FABP, in which one or more aminoacid residues have been altered (inserted, deleted and/or substituted)are also useful in the method and include, for example, substitutions ofCys in I-FABP at positions 27, 81, 82, 84, an Ala substitution atresidue 72 of I-FABP, and a Lys substitution at residue 27 of H-FABP.The FABP molecules may be fluorescently-labeled using a variety of knownlabels including but not limited to acrylodan, danzyl aziridine,4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazoleester (IANBDE), and4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole(IANBDA). Any label may be used in the practice of the invention as longas a measurable difference may be detected upon binding of a free fattyacid. For example, a difference in wavelength, signal intensity, orpolarization, or lifetime may be monitored. Further examples of labelswhich may be used include, but are not limited to, chromophores whichproduce a change in absorbance or optical activity and spin labels whichchange is detectable by electron spin resonance. In a preferredembodiment, a fluorescently-labeled FABP is acrylodan-derivedrecombinant rat intestinal fatty acid binding protein (referred to asADIFAB). Derivatization with acrylodan was performed using known methodssubstantially as previously described (U.S. Pat. No. 5,470,714 &Richieri, G. V, et al., J. Biol. Chem., (1992) 276: 23495-23501), andADIFAB is commercially available (FFA Sciences LLC, San Diego, Calif. ).Concentrations of FFA_(u) can be determined by the binding of theFFA_(u) to the fluorescently labeled fatty acid binding protein (FABP).A different fluorescence is exhibited when no FFA is bound to thefluorescently-labeled FABP. The concentration of FFA_(u) can bedetermined from the fluorescence difference. The wavelength emitted bythe fluorescently-labeled FABP depends upon the label and protein used.In one embodiment, the protein is either I-FABP or a I-FABP with asubstitution of Ala for Leu at position 72 where the label isacryolodan. These species are referred to as ADIFAB and ADIFAB2,respectively.

Briefly, ADIFAB2 was obtained as follows. First, a restriction fragment,carrying the Ala⁷² mutation and appropriate complementary ends, wassubstituted for the wild-type restriction fragment spanning the SalIsite at position 211 in the I-FABP cDNA sequence (Alpers, et al. (1984)Proc. Natl. Acad. Sci U.S.A. 81: 313-317) and a PmeI site at position291, which was introduced by site-specific mutagenesis. The mutatedrestriction fragment was constructed by annealing partiallycomplementary synthetic oligonucleotides carrying the desired sequence,“filling in” single-stranded regions with the DNA polymerase Klenowfragment, and digesting with SalI and PmeI to give the appropriatetermini. The Ala⁷² substituted I-FABP cDNA was inserted into the pET11vector and was expressed in the BL21(DE3) strain. The mutant I-FABP waspurified essentially by the method of Lowe, et al. (Lowe, et al. (1987)J. Biol. Chem. 262: pages 5931-5937) and yielded about 100 mg ofpurified protein per liter of Escherichia coli culture. Acrylodanderivatization was done as described previously for ADIFAB (see U.S.Pat. No. 5,470,714, incorporated herein by reference). Lipidex-5000chromatography was used to remove free acrylodan.

The binding affinities of ADIFAB2 have been found to be about 10-foldgreater than ADIFAB. The wavelength emitted by thesefluorescently-labeled FABP's is about 420 to 470 nm. The emissionwavelength for the FFA bound to the fluorescently-labeled FABP is about495 to 580 run. It will be understood that those skilled in the art canreadily substitute other fluorescently-labeled FABP in the disclosedassay.

The assay for determination of FFA_(u) levels in aqueous samplesmeasures the intensity of a shift in fluorescence from a firstwavelength, at which the derivatized FABP fluoresces when no FFA isbound, to a second wavelength, at which the derivatized FABP fluoresceswhen a molecule of FFA is bound, and the concentration of FFA_(u) isthen determined from the ratio (“R” value) of the two intensities offluorescence wavelengths as described in U.S. Pat. No. 5,470,714 andRichieri, et al. (1995) J. Lipid Research, vol. 36: pages 229-240, bothof which are incorporated herein by reference. Briefly, the ratio iscalculated using the following formula:

$R = \frac{{I(1)} - {{I(1)}{blank}}}{{I(2)} - {{I(2)}{blank}}}$wherein, I(1) and I(1)blank are the measured fluorescence intensitiesfor a sample containing ADIFAB or ADIFAB2 and a blank sample containingall reagents except ADIFAB or ADIFAB2, respectively at wavelength “1”;and 1(2) and I(2)blank are the corresponding fluorescence intensities atwavelength “2”. For ADIFAB, wavelength “1” is in preferably (but notabsolutely) in the range of 495 and 515 nm and wavelength “2” is in therange from 422 to 442 nm. Measurements of fluorescence intensities maybe obtained using standard techniques. As recognized by those skilled inthe art, under appropriate conditions, the Iblank intensities in theabove equation can be neglected.

Quantitative detection of levels of body fluid FFA_(u) that are elevatedover body fluid FFA_(u) levels found in normal healthy individuals canbe used to diagnose an ischemic condition. In one embodiment, anischemic condition is indicated by a concentration of unbound orwater-soluble free fatty acid in a body fluid sample that issignificantly higher than the concentration of unbound or water-solublefree fatty acid in a body fluid sample of a control population that doesnot have an ischemic condition. In one embodiment, an ischemic conditionis detected by levels of unbound or water-soluble free fatty acid in abody fluid sample that exceed the average normal levels of unbound orwater-soluble free fatty acid in a body fluid sample by about 2 standarddeviations. In another embodiment, unbound or water-soluble free fattyacid levels which are greater than about 5 nM are indicative of anischemic condition. In another embodiment, a level of unbound orwater-soluble free fatty acid in a body fluid sample greater than abouttwice the average value of unbound or water-soluble free fatty aciddetermined from a body fluid sample from a non-ischemic population isindicative of the ischemic condition. Elevated levels of unbound orwater-soluble free fatty acid in a body fluid sample which areconsiderably higher may also be detected.

The diagnostic method of the present invention is based on the discoverythat patients experiencing an ischemic condition have elevated levels oflipids such as FFA_(u) in body fluids such as blood, compared to normallevels of body fluid FFA_(u) in healthy individuals. While thediagnostic method may be carried out at any time, preferably, the testis carried out within 24 hours of the ischemic event. More preferably,the test is carried out within 10 hours of the ischemic event. Mostpreferably, the test is carried out within 3 hours of the ischemic eventso that treatment may be initiated. Many treatments for ischemicconditions such as cardiac ischemia must be initiated quickly (withinhours) in order to be effective. Treatment without a correct diagnosiscan be most deleterious to the patient. The ability to quickly diagnosean ischemic condition is an advantage of the invention described here.

Briefly, the FFA_(u) assay and determinations were performed as follows.Blood samples were diluted 100-fold in buffer (20 mMN-2-hydroxyethyl-piperazine-N′-2-ethane sulfonic acid (HEPES), 150 mMNaCl, 5 mM KCl and 1 mM Na₂HPO₄, adjusted to pH 7.4), yielding a serumalbumin concentration of about 6 μM. A solution of fatty acid-freealbumin plus ADIFAB or ADIFAB2 was the negative control. For each donor,two aliquots of serum were prepared: one “background” or “blank” sampleof 1% serum and one “experimental” sample of 1% serum plus ADIFAB orADIFAB2. The negative control, blank and experimental samples weremeasured at 22° C. or 37° C.

For each sample, multiple measurements of pairs of intensities werecollected at about 432 nm and 505 nm, for ADIFAB and about 450 and 550for ADIFAB2 and R values were determined after subtraction of theintensities of the blank sample. At least two separate measurements weredone on different days for each serum sample and the mean values andstandard deviations of FFA_(u) concentrations were determined. Todetermine the probabilities of a difference between sets of measures,differences in means were evaluated using Student's t test, where a pvalue of less than 0.05 was considered significant.

The present disclosed method for detecting ischemia may further comprisethe steps of taking measures to reduce the levels of ischemic markers.Also encompassed by the present disclosure is a method of determiningprognosis of a patient following treatment for ischemia by detectingischemia as disclosed herein and determining if the level of theischemic marker is trending towards the level of the marker in thenon-ischemic condition. The disclosed method also comprises theevaluation of the risks associated with elevated ischemic markers,either because the elevation of these markers reflects an underlyingpathologic state and/or because the ischemic markers are themselvesdeleterious.

EXAMPLE 1 [FFA_(u)] are Elevated Before Troponin I in PatientsPresenting to the Emergency Department (ED) with Different Forms ofMyocardial Ischemia

To determine the concentration of unbound free fatty acids [FFA_(u)] inpatients suffering from a wide range of “natural” ischemic insults wemeasured FFA_(u) levels in patients presenting to the emergencydepartment (ED) of the Hennepin County Medical Center (Minneapolis,Minn.). Samples were drawn at admission and several times later from 29patients. Each of these patients was diagnosed as having cardiacischemia, primarily by ST segment changes. The diagnoses for thesepatients included: acute myocardial infarction (AMI), congestive heartfailure (CHF), unstable angina, cardiac contusion, cardiac surgery,hypertension and cocaine induced ischemia. FFA_(u) were significantlyelevated in every patient diagnosed with cardiac ischemia, on average 20times higher than normals (this study also included 48 “normal” patientswhose average FFA_(u) levels were 3 nM).

FFA_(u) levels were elevated at the earliest draw (admission to the ED)in every patient, including the 9 AMI patients in this study (Table). Incontrast, 7 of these 9 AMI patients showed no elevation of the cardiacmarker Troponin I (TnI) at the time of admission. In these 7 patientsTnI became elevated between 4 and 32 hours after admission. Furthermore,most of the non-AMI patients did not reveal elevated TnI levels,consistent with [FFA_(u)] being a marker of ischemia before necrosis.

Another noteworthy feature of these results is that in the absence ofreperfusion therapy, FFA_(u) levels remain elevated for long periods (upto days) after the initial ischemic event (Table). Thus FFA_(u) respondrapidly to an ischemic event but return only slowly (presumably as theischemia resolves) to base line, thereby providing a uniquely widediagnostic window.

TABLE Time course of TnI and [FFA_(u)] in AMI^(a) TIME TnI [FFA_(u)]Patient (hours) (ng/ml) (nM) 1 0 0.3 314 4 0.8 27 2 0 0.3 23 54 7.1 46362 15.9 797 3 0 0.3 22 5 0.3 282 29 5.2 14 36 4.5 19 4 0 0.3 12 5 0.3 199 0.3 9 2.7 14 5 0 0.3 22 16 2.1 29 22 2.5 54 6 0 9 7 0.6 22 7 0 2. 8 103.1 112 8 0 0.4 18 13 1 19 9 0 10.6 7 7 6.3 11 21 6.9 36 27 5.4 25 384.2 26 50 2.1 10 ^(a)Hennepin AMI patients. Time is hours from time ofadmission. Baseline TnI values were 0.3 ng/ml and normal [FFA_(u)] were3 nM. Two or more serial measurements were done for each patient(separated by thick lines).

These results indicate the potential power of the FFA_(u) assay; aserious ischemic event can be detected hours to days before currentcardiac markers indicate disease, raising the possibility of earlierintervention and significant improvement in patient outcome.

EXAMPLE 2 Results from the Thrombolysis in Myocardial Infarction II(TIMI-II) Trial Indicate High Sensitivity and Specificity for Detectionof AMI using [FFA_(u)] Measurements

To obtain more detailed information about the FFA_(u) response in acutemyocardial infarction, we used blood specimens from the TIMI II trial(TIMI Study Group, New England Journal of Medicine (1989) vol. 320, page618-625). These specimens have been maintained by the National HeartLung Blood Institute and were made available for our investigations ofFFA_(u) levels in ischemia. Patients were enrolled in the TIMI II trialif they presented to an ED with chest pain within 4 hours of the initialsymptoms and exhibited an ST segment elevation. Every patient wastreated intravenously over 6 hours with either 100 mg (90.5% of the 3262patients treated) or 150 mg (9.5% of the 3262 patients treated) t-PAstarting within about 10 minutes of enrollment. Blood samples were drawnupon admission (just before t-PA), 50 minutes after t-PA, and then 5 hand 8 h after start of t-PA treatment. A set of 4 such samples wasstored at −70° C. for each of approximately 2,500 patients. For eachpatient, the TIMI investigators recorded about 825 clinical, physicaland chemical observations.

Results for the first 500 patients provide important insights into therelationship between acute cardiac ischemia and FFA_(u) levels. [Sampleswere received in individual shipments of 1000 samples (250 patients).FIGS. 1 and 2 show results for shipments 1 and 2, respectively.](Kleinfeld, et al. (2002) J. Am. Coll. Cardiol. 39: 312A).

Upon admission the average FFA_(u) (ADIFAB2) value was 13 nM, more than14 standard deviations above the normal value (2.8±0.7 nM). This resultdemonstrates clearly that [FFA_(u)] are highly elevated, early in theischemic event. Using a 5 nM cutoff, the predicted sensitivity fordetection of AMI was 91% using FFA_(u) at admission (using FFA_(u) atadmission and at 50 minutes increases sensitivity to >98%). The resultsshow that >98% of the patients have FFA_(u) levels >3 standard deviationabove normal levels when the first two time points are included (>91%with only 1^(st) time point).

At the same time, CK values at time 0 are generally less than the upperlimit of normal (FIG. 3). Creatine kinase levels at admission wereelevated in less than 19% of the patients and even in these patients theincreases were modest, on average <50% above the upper limit of normalfor the test. Not until 4 h post admission were creatine kinase levelselevated substantially in a majority of the patients.

At 50 minutes the average FFA_(u) (22 nM) is substantially higher thanthe already elevated initial values. These samples were obtained about50 minutes after administration of t-PA (FIGS. 1 and 2). Thesignificantly higher average FFA_(u) levels may reflect reperfusioneffects and the ischemia mediated increases in [FFA_(u)] with increasingtime. In any event, this time course appears to be changed abruptly byt-PA treatment because FFA_(u) levels at 5 and 8 hours revealsignificant decreases following reperfusion; the average value at 8hours is significantly (p<0.05) lower than the FFA_(u) level at time ofadmission. This biphasic response appears to be correlated withreperfusion because the AMI patients in the Hennepin study who did notreceive reperfusion therapy reveal [FFA_(u)] that remained elevated fortimes greater than 24 h (data not shown). These findings, that FFA_(u)may increase in response to reperfusion and then decrease within 5 h,indicate that FFA_(u) levels might be used to evaluate the success ofreperfusion therapy.

The [FFA_(u)] response of these patients appears to be an intrinsic andindependent property of the pathology. FIGS. 1 and 2 reveal thatmeasurements of the [FFA_(u)] distributions at each of the 4 differentsample times, yield virtually identical parameters for the distributionsof 2 different sets of 250 patients; samples in shipments 1 and 2 arefor two sets of different patients. Moreover, almost all patients withconfirmed MIs revealed FFA_(u) (50 minutes)>[FFA_(u)] (admission),whereas for most of the approximately 3.3% of patients without confirmedMI, [FFA_(u)] (50 minutes)≦[FFA_(u)] (admission).

EXAMPLE 3 High Levels of [FFA_(u)] in TIMI-II Trial Samples at Time ofAdmission Correlate with Higher Mortality 3 Years Post Admission

[FFA_(u)] values at the time of admission are correlated with a morethan 2-fold increase in mortality. [FFA_(u)] values for specimens drawnat time of admission (about 400 of the 500 patients) were sorted andpartitioned into quartiles. The number of deaths, by any cause up toabout 3 years after admission, were counted for each quartile. Theprimary (75%) cause of death was cardiovascular disease. The resultsshow a more than 2 fold increase in death rate from lowest to highestquartile with a p value <0.025 (FIG. 4). In contrast, the cardiacmarker, creatine kinase, was normal at the time of admission for most(>80%) of these patients (FIG. 3). Furthermore, [FFA_(u)] values werenot correlated with age, gender, race, weight, other disease,medications or systolic blood pressure on admission. These resultsindicate that in addition to their other diagnostic characteristics,FFA_(u) levels measured at time of admission to the ED can be used tostratify patients according to their mortality risk (Kleinfeld, et al.(2002) J. Am. Coll. Cardiol. 39: 312A).

EXAMPLE 4 High Levels of [FFA_(u)] in TIMI-II Trial Samples at Time ofAdmission Correlate with Increased Rate of Severe Hemorrhage After t-PATherapy

[FFA_(u)] at admission also predict increased rates of severehemorrhagic events after t-PA therapy. A similar analysis as formortality (Example 3) was performed for severe hemorrhagic events. Thenumber of patients that experienced severe hemorrhagic events wasdetermined for each quartile (FIG. 5). The results reveal a more than 3fold increase in the rate of severe hemorrhagic events from lowest tohighest quartile of the distribution of [FFA_(u)] values at admission(before the start of t-PA administration) with p<0.01. One of thepossibilities raised by these results is that rates of hemorrhagemediated by t-PA might be reduced if FFA_(u) levels were lowered.

Although the results of FIGS. 4 and 5 do not indicate whether elevatedFFA_(u) levels directly and independently contribute to increased ratesof death and hemorrhage, abundant in vitro work demonstrates thatelevated FFA are potent perturbers of many cellular functions. Inaddition, clinical evidence for a direct role of FFA is stronglysuggested by many studies pointing to their arrhythmogenic role andtheir potential role in cardiovascular disease. Results from our TIMI IItrial also suggest that [FFA_(u)] are a strong independent factor ofadverse outcomes. Thus, [FFA_(u)] do not simply reflect the demographicsof this patient population; no correlation was observed with age,gender, race, or weight, for example. Moreover, the correlations of[FFA_(u)] at admission with mortality and hemorrhagic events, appear tobe independent; although the mortality rate was higher for patients whoexperienced severe hemorrhagic events (22% vs 11% for all patients),only 15% of deaths were due to hemorrhage.

EXAMPLE 5 Blood Levels of FFA in Ischemic Patients do not Originate fromthe Ischemic Tissue

Cerebral and myocardial ischemia in humans result in increased plasmaFFA levels by more than 7 fold above normal (Kleinfeld, et al. (1996)Amer. J. Cardiol. 78: 1350-1354; Kurien, et al. (1966) The Lancet 16:122-127; Oliver, et al. (1994) The Lancet 343: 155-158). It is also wellknown that FFA accumulate within the cells of an ischemic tissue, atleast for isolated organs (Bazan, et al. (1970) Biochim. Biophys Acta218: 1-10; Van der Vusse, et al. (1997) Prostaglandins, Leukotrienes andEssential Fatty Acids 57: 85-93). However, this generation of FFA byischemic tissue cannot account for the increases in plasma FFA observedin ischemia. This follows because the amount of FFA generated by theischemic tissue, even under conditions of complete ischemia of the wholeorgan, is negligible compared to normal plasma FFA levels, let alone themore than 7 fold increases observed in ischemic patients. For example,under normal conditions plasma FFA turns over every 2 min. which at(normal) plasma total [FFA] of about 500 μM translates into about 0.2 gof FFA/min. The FFA produced in ischemic tissue derives fromphospholipid (Bazan, et al. (1970) Biochim. Biophys Acta 218: 1-10;Goto, et al. (1988) Stroke 19: 728-735; Jones et al. (1989) Am. J.Pathol. 135: 541-556), which comprises less than 1% by (wet) weight of atypical (non-adipose) cell. Therefore, under ischemic conditions, wheretotal FFA is 2 to 7 fold greater than the normal level, more than 20g/min. of tissue would have to be used to maintain the observed levelsof FFA. This is not compatible with life and indicates that increases inplasma FFA are derived from non-ischemic tissue.

In the specific example of complete cerebral ischemia (decapitation) theamount of total FFA that accumulates in rat brain over a period of 30minutes is about 0.8 μmoles (Ikeda, et al. (1986) J. Neurochem. 47:123-132). The total amount of plasma FFA in a 300 g rat is greater than5 μmoles (>10 ml plasma of 500 μM FFA). In a rat model of reversiblemiddle cerebral artery ischemia (a much less profound ischemic insultthan decapitation), total plasma FFA increases about 2 fold andtherefore an additional 5 μmoles of FFA is added to plasma every 2minutes or about 75 μmoles in 30 minutes. Thus the 0.8 μmoles producedin 30 minutes of complete ischemia is negligible in comparison to theamount of FFA needed to raise plasma levels to those observed inischemia. The most likely source of tissue with sufficient capacity togenerate the large quantities of FFA observed in cerebral ischemia isadipose tissue. We have found that within minutes of cerebral ischemiaincreases are observed in plasma levels of TNF_(α), a potent activatorof adipose lipolysis (Ryden, et al (2002) J. Biol. Chem. 277:1085-1091)and that this TNF_(α) is well correlated with FFA_(u) increases inplasma.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

1. A method of detecting an ischemic condition in a mammal comprisingthe steps of: (a) measuring a level of an ischemic marker which is anunbound or water-soluble lipid in a test sample from a fluid of saidmammal; (b) comparing the measured level of said ischemic marker and apredetermined value for the level of said marker; and (c) determining ifthe level of said ischemic marker measured in said test samplecorrelates with an ischemic condition in said mammal, wherein saidpredetermined value for the level of said marker is indicative of thenon-ischemic condition and wherein the ischemic marker measured issoluble in an aqueous buffer.
 2. The method according to claim 1,wherein the mammal is a human.
 3. The method according to claim 2,wherein the fluid is selected from the group consisting of blood, serum,plasma, saliva, bile, gastric juices, cerebral spinal fluid, lymph,interstitial fluid and urine.
 4. The method according to claim 3,wherein the fluid is blood.
 5. The method according to claim 1, whereinthe lipid is a fatty acid.
 6. The method according to claim 1, wherein aheart is ischemic.
 7. The method according to claim 1, wherein theischemic marker is detected by UV/VIS, infrared, microwave, radio,absorption or fluorescence spectroscopy.
 8. The method of claim 1,further comprising an initial step of selecting a mammal presentingsymptoms of ischemia.
 9. The method of claim 1, further comprising thestep of administering anti-ischemic therapy if the level of ischemicmarker correlates with ischemia in said mammal.
 10. The method of claim9, wherein the anti-ischemic therapy comprises a means to lower levelsof serum fatty acids.
 11. The method of claim 9, wherein theanti-ischemic therapy is reperfusion therapy, antithrombolytic therapy,angiogenic therapy or surgery.
 12. The method of determining ischemiaaccording to claim 1, wherein the ischemic marker is an unbound orwater-soluble free fatty acid.
 13. The method according to claim 12,wherein the unbound or water-soluble free fatty acid is detected by aprotein that binds fatty acid.
 14. The method according to claim 13,wherein said protein that binds an unbound or water-soluble free fattyacid is fluorescent and exhibits a fluorescence that is different whenthe fatty acid is bound than when it is not bound.
 15. The methodaccording to claim 14, wherein the protein is one of the family ofintracellular Fatty Acid Binding Proteins (FABPs) that have molecularweights between about 13,000 and 16,000 Dalton.
 16. The method accordingto claim 15, wherein the FABP is a rat intestinal FABP.
 17. The methodaccording to claim 16, wherein the FABP is ADIFAB.
 18. The methodaccording to claim 17, wherein the acrylodan-labeled FABP is ADIFAB2.19. The method according to claim 13, wherein the protein is albumin.20. The method according to claim 19, wherein the albumin is covalentlylabeled with 7-hydroxycoumarin or anthraniloyl.
 21. The method accordingto claim 12, wherein a level of the unbound or water-soluble free fattyacid greater than 2 standard deviation units above an average value of alevel of the unbound or water-soluble free fatty acid determined from anon-ischemic population is indicative of the ischemic condition.
 22. Themethod according to claim 12, wherein a level of the unbound orwater-soluble free fatty acid greater than about twice an average valueof a level of the unbound or water-soluble free fatty acid determinedfrom a non-ischemic population is indicative of the ischemic condition.23. The method according to claim 12, wherein a level of the unbound orwater-soluble free fatty acid greater than about 2.5 nM is indicative ofthe ischemic condition.
 24. The method of determining ischemia accordingto claim 1, wherein the ischemic marker is total unbound free fattyacid.
 25. The method according to claim 24, wherein a level of the totalunbound free fatty acid greater than 2 standard deviation units above anaverage value for the level of the total free fatty acid determined froma non-ischemic population is indicative of the ischemic condition. 26.The method according to claim 24, wherein a level of the total unboundfree fatty acid greater than about twice an average value of a level ofthe total free fatty acid determined from a non-ischemic population isindicative of the ischemic condition.
 27. The method according to claim24, wherein the ischemic marker is a ratio of total unbound free fattyacid to albumin.
 28. The method according to claim 27, wherein the ratioof total unbound free fatty acid to albumin greater than about twostandard deviation units above an average value of the ratio of totalfree fatty acid to albumin determined from a non-ischemic population isindicative of the ischemic condition.
 29. The method according to claim27, wherein the ratio of total unbound free fatty acid to albumingreater than about twice an average value of the ratio of total freefatty acid to albumin determined from a non-ischemic population isindicative of the ischemic condition.